Actinobacillus pleuropneumoniae (App) culture supernatant antiviral effect against porcine reproductive and respiratory syndrome virus (PRRSV) occurs prior to the viral genome replication and transcription through actin depolymerization.
Yenney Hernandez Reyes1, Chantale Provost1, Carolina Kist Traesel1, Mario Jacques1, Carl A. Gagnon1*
1Centre de recherche en infectiologie porcine et avicole (CRIPA) et Groupe de recherche sur les maladies infectieuses en production animale (GREMIP), Faculté de médecine vétérinaire,
Université de Montréal, St-Hyacinthe, Québec, Canada J2S 2M2
* Corresponding author: Carl A. Gagnon, Faculté de médecine vétérinaire, Université de Montréal, 3200 rue Sicotte, St-Hyacinthe, Québec, Canada, J2S 2M2. email: carl.a.gagnon@umontreal.ca 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
ABSTRACT
Purpose. Recently, a strong antiviral activity of App culture supernatant against PRRSV was discovered. Following this finding, the objective of the present study was to understand how App culture supernatant inhibits PRRSV replication in its natural targeted host cells, i.e. porcine alveolar macrophages (PAM).
Methodology. Several assays were conducted with App culture supernatant treated PRRSV infected cell lines such as PAM, SJPL and MARC-145 cells. RT-qPCR assays were used to determine the expression levels of type I and II interferons mRNAs, viral genomic (gRNA) and sub-genomic RNAs (sgRNAs). Proteomic, Western blot and immunofluorescence assays were conducted to determine the involvement of actin filaments into the App culture supernatant antiviral effect.
Results/Key findings. Type I and II interferons mRNAs expression were not upregulated by
App culture supernatant. Time courses of gRNA and sgRNAs expression levels demonstrated
that App culture supernatant inhibits PRRSV infection before the first viral transcription cycle. Western blot experiments confirmed an increase in the expression of cofilin (actin cytoskeleton dynamics regulator) and immunofluorescence also demonstrated a significant decrease of actin filaments in App culture supernatant treated PRRSV infected PAM cells. App culture supernatant antiviral activity was also demonstrated against other PRRSV strains of genotypes I and II.
Conclusion. App culture supernatant antiviral effect against PRRSV take place early during PRRSV infection. Results suggest that App culture supernatant antiviral effect may take place via the activation of cofilin, which induces actin depolymerization and subsequently, probably 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39
Keywords: Porcine reproductive and respiratory syndrome virus; PRRSV, Actinobacillus
pleuropneumoniae; App; antiviral effect; actin; cofilin; virus replication; porcine alveolar
macrophages; PAM cells. 41
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INTRODUCTION
Porcine reproductive and respiratory syndrome (PRRS) is a worldwide endemic disease, which causes significant economic losses in pig-producing countries. The causative agent, PRRS virus (PRRSV), belongs to the family Arteriviridae of the Nidovirales order. Following the discovery of several new arteriviruses, a new taxonomic classification was recently proposed [1]. PRRSV is an enveloped single-stranded positive-sense RNA virus of approximately 15 kb in length that encodes for at least 11 open reading frames (ORFs) [2]. It has a strongly restricted cell tropism for the monocyte–macrophage lineage in vivo. In fact, in its natural host, the primary cells targeted by PRRSV are the fully differentiated porcine alveolar macrophages (PAM), which are often used for in vitro study [3-6]. The only two continuous cell line non-genetically modified able to fully replicate PRRSV are: African green monkey kidney cell line MA-104 and its derivatives like MARC-145 [7] and, the newly permissive reported St-Jude porcine lung (SJPL) cells, which were found to be of monkey origin [8, 9].
Following PRRSV entry and release of its viral genome into the cytoplasm, the PRRSV ORF1 is translated. Then, the resulting non-structural proteins trigger the formation of the replication-transcription complex, which associates with double membrane vesicles to initiate genome replication and transcription process [10-12]. The genome replication is produced by the continuous synthesis of negative (-) full-length RNA strands using as template the positive genomic RNA [(+) gRNA]. Then, the (-) RNA strands will lead to the formation of new (+) gRNAs [13]. The genome transcription process conducts to the synthesis of a nested set of sub-genomic mRNAs (sg mRNAs). According to a model proposed by Sawicki and collaborators [14], the generation of these sg mRNAs comes from a discontinuous RNA synthesis process, 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
where (-) sg RNA strands are produced and subsequently are used as templates for the synthesis of the sg mRNAs.
Current management strategies, which focus on the prevention of PRRSV infection (ex. biosecurity measures, surveillance, whole herd depopulation and repopulation, and herd closure [15]) and vaccination using commercially available modified live-attenuated vaccines or autogenous killed vaccines, have mostly been demonstrated to be inadequate for long-term control of PRRS [16]. This prompts the search of novel strategies to control PRRSV infection. In this sense, recent published works have discovered natural compounds with antiviral activities against PRRSV such as macrolides [17], N-acetylpenicillamine [18], Cryptoporus volvatus extract [19], morpholino oligomer [16, 20], Matrine [21], sodium tanshinone IIA sulfonate [22], and flavaspidic acid AB [23]. Each of these compounds inhibits PRRSV replication differently. For instance, the flavaspidic acid AB inhibits PRRSV internalization and cell-to-cell virus transmission, probably by the induction of type I interferons (IFN) [23]. Sun and colleagues demonstrated that Matrine inhibits N protein expression and has anti-apoptotic functions [21]. Moreover, Cryptoporus volvatus extract was demonstrated to inhibit PRRSV infection in vitro and in vivo, probably by the direct inhibition of PRRSV polymerase activity [19]. However and despite these efforts, there are no effective commercially available drugs to prevent PRRSV infection.
Our previous work revealed that the cell culture supernatant of Actinobacillus pleuropneumoniae (App) mutant strain (AppΔapxICΔapxIIC) possesses an antiviral activity against PRRSV in infected SJPL and PAM cells, but this antiviral activity was not observed in infected MARC-145 cells [24]. It was the first time that a bacterial antiviral effect against PRRSV in vitro was reported. Thereafter, our team has investigated the mechanisms involved in the antiviral activity 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89
displayed by App culture supernatant. It was found that App culture supernatant was able to induce cell cycle arrest at the G2/M-phase in PRRSV infected SJPL cells such as two other G2/M-phase cell cycle inhibitors, which are also inhibiting PRRSV replication [25]. Unfortunately, during our previous investigation, the effect of App culture supernatant on PAM cell cycle could not be investigated because unlike SJPL cells, the PAM cells do not replicate. Therefore, it is crucial to investigate the antiviral activity displayed by App culture supernatant in a more pathogenic relevant model, i.e. PRRSV infected PAM cells. Results showed that App culture supernatant blocks PRRSV replication prior its first cycle of genome replication and transcription in infected PAM and SJPL cells. Following proteomic analyses, data suggest that the early App culture supernatant antiviral effect against PRRSV in infected PAM cells take place via the activation of cofilin and thus actin depolymerization, which probably affect PRRSV endocytosis. 90 91 92 93 94 95 96 97 98 99 100 101 102
MATERIAL AND METHODS
Cells
The MARC-145 and SJPL cell lines were maintained as previously described [24]. The SJPL cell line was kindly provided by Dr. R.G. Webster) [8]. PAM cells were obtained from lungs of 2 to 14 week old pigs as previously described [9, 24]. PAM cells were cultured for 24 hrs in complete Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, GibcoBRL) complemented with 10% fetal bovine serum, 2 mM L-glutamine, 0,1 mM HEPES, 1 µM Non-essential amino acids, 250 g/L Amphotericin B, 10 units/mL penicillin, 10 µg/mL streptomycin and 100 mg/L gentamicin (Wisent) [24]. All cells were cultured and infected at 37°C in 5% CO2 atmosphere.
Viral and bacterial strains
The PRRSV strain used in this study was the Canadian genotype II reference strain IAF-Klop and the virus stocks were obtained as previously described [9]. The App strain used in this study was the mutant MBHPP147 (AppΔapxICΔapxIIC) from the strain S4074, which is a serotype 1 reference strain. This mutant produces non-active ApxI and ApxII toxins and was kindly provided by Ruud P.A.M. Segers (MSD Animal Health). App strain was cultured on brain heart infusion (BHI) broth and/or agar (Oxoid) supplemented with 15 µg/mL nicotinamide adenine dinucleotide (NAD) at 37°C in 5% CO2. The App culture supernatant was obtained as previously described [24]. 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125
Infection of cells
Cells were infected, as previously described [24], with PRRSV IAF-Klop strain at 0.5 MOI and incubated in DMEM without serum or other additives during four hours (hrs), then a soft washing step using phosphate buffer saline solution (PBS) was done. Thereafter, the App culture supernatant or complete medium in the case of controls were added (protocol #1). Another infection protocol was tested to determine if App culture supernatant added prior to PRRSV infection had an impact on the results (protocol #2). Cells were pre-treated with the bacterial supernatant (or medium for controls) during two hrs, followed by PRRSV infection at 0.5 MOI in DMEM without serum or other additives for four hours, then a soft wash step with PBS was done and finally, App culture supernatant or the complete medium were added. Both infection protocols were used throughout all this study unless specified.
Cells viability and mortality
2x105 PAM cells/well were incubated during 24 hrs. Afterwards, cells were infected with PRRSV using the protocol #1 and incubated in the presence of App culture supernatant or complete medium during 48 hrs. Cell viability was measured with CellTiter 96® Aqueous One Solution Cell Proliferation Assay (Promega) at 52 hrs pi. Cellular mortality was determined using the lactate dehydrogenase (LDH)-measuring CytoTox 96® nonradioactive cytotoxicity assay (Promega). Mechanically lysed cells were used as 100% mortality positive control. For both methods, non-infected cells were used as a negative control and the absorbance was measured at 490 nm with a SynergyTM HT multi-detection microplate reader (Biotek). Both tests were repeated three times.
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Type I IFN, IFN gamma (IFN-γ) and β-actin relative mRNAs expression.
4x106 PAM cells/well and 5x105 SJPL or MARC-145 cells/well were infected with PRRSV and incubated with or without App culture supernatant or complete medium during 48 hrs. As a positive control for innate immunity induction, PAM were transfected with Polyinosinic– polycytidylic acid potassium salt (Poly I:C) [50 μg/mL] (Sigma-Aldrich), using polyethyleneimine (PEI) [1 μg/μL] (Sigma-Aldrich). Total cellular RNA was extracted from cells using Trizol reagent (Invitrogen). RNA quantification was performed using NanoDrop® ND-1000 (NanoDrop Technologies). One μg of total RNA was reverse-transcribed using the QuantiTect reverse transcription kit (Qiagen). Specific cDNA targets were amplified using the SsoFast™ EvaGreenW Supermix kit (Bio-rad) in the Bio-Rad CFX-96 sequence detector apparatus. The PCR amplification program for all cDNA targets consisted of an enzyme activation step of 3 min at 98°C, followed by 40 cycles of a denaturing step for 2 secs at 98°C and an annealing/extension step for 5 secs at 57°C. The primer pairs used for the amplification of type I IFNs and IFN-γ in PAM were: IFN-α: F 5’-ACTCCATCCTGGCTGTGAGGAAAT-3’
and R 5’-TCTGTCTTGCAGGTTTGTGGAGGA-3’; IFN-β: F
CTCTCCTGATGTGTTTCTCC-3’ and R GTTCATCCTATCTTCGAGGC-3’; IFN- γ: F 5’-GAGCCAAATTGTCTCCTTCTAC-3’ and R 5’- CGAAGTCATTCAGTTTCCCAG-3’. The porcine β-actin and monkey β-actin genes amplification were performed using the primers F 5’-ACCACTGGCATTGTCATGGACTCT-3’ and R 5’-ATCTTCATGAGGTAGTCGGTCAGG-3’; and F 5’-GGCATCCATGAAACTACCTTC-3’ and R 5’-AGGGCAGTAATCTCCTTCTG-3’, respectively. Peptidylprolyl isomerase A (PPIA) and beta-2 microglobulin (B2M) were used as normalizing genes in PAM and SJPL/MARC-145 cells, respectively, and were amplified using the following primers pairs: PPIA: F 5’-TGCAGACAAAGTTCCAAAGACAG-3’ and R 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171
5’-GCCACCAGTGCCATTATGG-3’; B2M [9]: F 5’-GTGCTATCTCCACGTTTGAG-3’ and R 5’-GCTTCGAGTGCAAGAGATTG-3’. Primer sequences were designed from the NCBI GenBank mRNA sequences using web-based software primerquest (Integrated DNA technologies). Uninfected cells were used as the calibrator reference in the analysis. Differences in quantification between groups were calculated using the 2-ΔΔCt method. These experiments were repeated three times in duplicate.
PRRSV genome replication/transcription kinetics
4x106 PAM cells/well and 5x105 SJPL or MARC-145 cells/well were incubated during 24 hrs, thereafter, were infected with PRRSV with or without App culture supernatant. At different times post-infection (pi), samples were collected to perform a specific RT-qPCR assay. The strategy used to determine PRRSV genome replication and transcription have already been published, however, new primers were designed for this study [26]. Briefly, total RNAs were extracted from cells and quantified as described above. 1.5 µg of total RNA was reverse-transcribed using M-MLV reverse transcriptase (Invitrogen). Two pmol of gene-specific reverse primers, R 5’-AGAAAGCACGTAAGCTCCAGCCAA-3’ for the detection of only the PRRSV (+) gRNA (which targets PRRSV ORF1 gene) and R 5’-AGCATCTGGCACAGCTGATTGACT-3’ for the detection of viral sg mRNAs (which targets PRRSV ORF7 gene) were used. It is important to specify that with ORF7 reverse primer, all the (+ strand) ORF7 sequences are detected, which include the ORF7 sequence from PRRSV viral genome and all the viral sg mRNAs sequences, explaining its use to quantify genome transcription. The obtained cDNA was treated with 1.5 μg RNase A (Invitrogen) for 30 minutes at 37°C to remove the remaining RNAs, followed by inactivation of RNase A by heating at 95°C for 10 minutes. Two µl of cDNA was amplified 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194
using the same reagents and conditions described above. The primer pairs used for PRRSV ORF1 amplification was F TGTGAGTTTGACTCGCCAGAGTGT-3’ and R TACAGTCTGCAACAATGCCAAGCC-3’; and for PRRSV ORF7 was F 5’-GCGGCAAGTGATAACCACGCATTT-3’ and R 5’-TGCTGCTTGCCGTTGTTATTTGGC-3’. The Ct values obtained were transformed in PRRSV (+) gRNA and PRRSV sg mRNAs copies/mL, for PRRSV genome replication and transcription quantification, respectively, based on a generated standard curve. First, PRRSV viral genome molecular weight was calculated using PRRSV ATCC VR2332 reference strain complete viral genome sequence (GenBank accession number: EF536003) and a formula available in Life technologies web site ( http://www.lifetechnologies.com/ca/en/home/references/ambion-tech-support/rna-tools-and-calculators/dna-and-rna-molecular-weights-and-conversions/). Afterwards, PRRSV viral genome was purified from our virus stock and its concentration was determined. Then, the amount of PRRSV viral genome copies was calculated. Thereafter, 10-fold serial dilutions of the PRRSV virus stock purified RNA was realized and RT-qPCR assays were performed as described above to establish the standard curve. All experiments were repeated three times in duplicate. As internal controls, RT-qPCR for the mRNA detection of PPIA (in PAM cells) and B2M (in SJPL and MARC-145 cells) housekeeping genes was also performed on the same RNA sample preparations using the primers described above.
Western blot assays
4x106 PAM and 5x105 SJPL/MARC-145 cells were infected and incubated with or without App culture supernatant. At 52 hrs pi, 40 μg of total proteins from each sample were loaded and were fractionated by electrophoresis on 10% (w/v) SDS-PAGE gels, then transferred onto a 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217
nitrocellulose membrane (Bio-rad) using Trans-Blot® SD Semi-Dry Transfer Cell (Bio-rad). Membranes were blocked with TBS-Tween 20 solution containing 5% (w/v) BSA (Sigma-Aldrich) or 5% (w/v) non-fat dry milk during 2-3 hrs at room temperature. Subsequently, they were labeled with a 1:1,000 dilution of rabbit cofilin antibody (# 3312, Cell Signaling Technology) and with a 1:2,500 dilution of mouse monoclonal β-actin antibody (mAbcam 8226, Abcam) and incubated at 4°C overnight. Horseradish peroxidase-conjugated goat anti-rabbit IgG and horseradish peroxidase-conjugated goat anti-mouse (Thermo Scientific) at a dilution of 1:3,000 were used as secondary antibodies. The protein bands were visualized using the SuperSignal® West Dura Extended Duration Substrate (Thermo Scientific) in the FUSION-FX Chemiluminescence System (Montreal Biotech). The same membranes were mildly striped using the protocol described in Abcam website (http://www.abcam.com/index.html? pageconfig=resource&rid=11353&source=pagetrap&viapagetrap=strippingforreprobing) and were re-probed with rabbit GAPDH monoclonal antibody (#5174) to confirm equal loading and with the rabbit phospho-cofilin antibody (#3311, Cell Signaling Technology), both at a dilution of 1:1,000. All experiments were repeated at least two times.
Immunofluorescence assay (IFA) for the detection of PRRSV antigen and F-actin
Cells were fixed at 52 hrs pi, during 30 minutes at room temperature, with a 4% paraformaldehyde (PFA) solution prepared as described previously [9]. Uninfected cells were used as negative control. The IFA assay was performed as described previously [9]. Briefly, the fixed cells were washed with PBS and were permeabilized during 10 minutes with a PBS solution containing 1% Triton X-100. Subsequently, the cells were washed with PBS-Tween 20 (0.02%) and incubated for 30 minutes with PBS containing 0.2% Tween 20 and 1% BSA. Then, 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240
the cells were incubated with the α7 rabbit monospecific antiserum (a specific anti-N PRRSV protein antibody) diluted 1/200 at 4°C overnight [27]. Finally, the cells were washed and incubated in 1/160 dilution of anti-rabbit specific antiserum FITC conjugated (Sigma-Aldrich) and in 1/40 dilution of Alexa Fluor® 594 phalloidin (a high-affinity F-actin probe conjugated) (Invitrogen) during 30 minutes at room temperature. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich). PAM stained cells were visualized by confocal laser scanning microscopy (Olympus FV1000 IX81). MARC-145 and SJPL cells were visualized using a DMI 4000B reverse fluorescence microscope (Leica Microsystems). Images of these cells were taking with a DFC 490 digital camera (Leica Microsystems).
PRRSV replication in the presence of cytochalasin D
2x105 PAM, and 1x104 MARC-145 and SJPL cells were infected using protocols #1 and #2 but the App culture supernatant was replaced by 3µM of cytochalasin D (Sigma-Aldrich). Three µM of cytochalasin D were used to make sure that PRRSV replication will be inhibited because lower quantities (1-2µM) were previously reported to inhibit PRRSV infection [28]. At 52 hrs pi, the production of PRRSV infectious particles were quantified by the Kärber method as previously described [9, 24].
App culture supernatant antiviral activity against other PRRSV viruses
PRRSV strains used in this experiment were PRRSV genotype I reference strain Lelystad virus (LV), and two others genotypes II pathogenic strains (FMV09-1155278 and FMV13-LMIVV). Thus, 1x104 cellswere infected with each virus using the protocol #1. The infectious dose of 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262
each virus was calculated as described above. All experiments were repeated three times in duplicate.
Statistical analyses
A One-way ANOVA model, followed by Tukey’s Multiple Comparison Test (GraphPad Prism Version 5.03 software) was used to establish if statistically significant differences existed between infected or uninfected cells treated or not with the App culture supernatant in the viability and mortality tests. The same test was used to determine whether the viral titers in the presence or absence of cytochalasin D were statistically significant. This statistical test was also used to determine whether App culture supernatant may modulate the expression of F-actin in treated and PRRSV infected cells. Two-way ANOVA model, followed by Bonferroni post-hoc tests (GraphPad Prism) was performed to determine whether there was a difference in PRRSV replication/transcription between App culture supernatant treated cells and untreated cells. Several t tests statistical analyses (unpaired t-tests) were also performed with PRRSV replication/transcription kinetic results at 4 hrs pi. The same test was also used to determine if type I and type II IFN relative expressions were statistically different in App culture supernatant treated cells compared to untreated cells. Moreover, t-tests statistical analyses were also used to determine whether App culture supernatant may modulate the -actin mRNA expression in treated and PRRSV infected cells compared to control cells. To establish if App culture supernatant possesses an antiviral effect against different PRRSV strains, a two-way ANOVA model, followed by Bonferroni post-hoc tests and t-tests (unpaired) statistical analyses were also performed. Differences between experimental groups were considered statistically significant with a P<0.05. 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285
RESULTS
Impact of App culture supernatant on cells viability and mortality
The viability test showed no statistically significant differences between the negative control and the App culture supernatant treated cells (Figure 1A). PRRSV infected cells had the lowest cell survival compared to all the other treatments. PRRSV infected PAM cells treated with App culture supernatant had a significantly higher cell survival (OD: 1.39 ± 0.27) compared to App culture supernatant non-treated PRRSV infected cells (OD: 1.03 ± 0.21). Opposite results were obtained with the mortality test (Figure 1B). In fact, when PRRSV infected cells were treated with App culture supernatant, it was improving significantly the mortality rate of the cells (43.12% ± 3.23) compared to PRRSV infected and untreated cells (59.19% ± 3.04). In resume, both methods showed that App culture supernatant did not affect PAM cells integrity and metabolism and had a positive effect on PAM cells survival when they are PRRSV infected.
Type I IFN and IFN-γ mRNAs relative expression in App culture supernatant treated cells Specific RT-qPCR tests were performed to determine if the App culture supernatant induces the expression of type I and II IFN mRNAs in PAM cells because INF are known to be potent antiviral molecules against PRRSV infection [29-31]. App culture supernatant only induced a small level of IFN- mRNAs expression, which was slightly increased compared to non-infected and non-treated PAM cells and had no modulation effect on IFN-β and IFN- mRNAs expression levels (Figure 2). PRRSV induced a significant increase in IFN-α and IFN-β mRNA relative expressions, i.e. 8.52 ± 3.74 and 41946 ± 37548 times more, respectively, compared to non-infected/non-treated cells (Figures 2A and 2B). Significant decreases in IFN-α and IFN-β mRNA relative expressions were observed in infected PAM cells treated with the bacterial 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308
supernatant (2.05 ± 0.36 and 1100 ± 2260, respectively) compared to PRRSV infected cells alone indicating that App culture supernatant had a major impact on PRRSV infected PAM cells (Figures 2A and 2B).
PRRSV genome replication and transcription kinetics in the presence of App culture supernatant
In PRRSV infected PAM cells, starting between 8 and 16 hrs post-infection (pi), an increase in (+) gRNA copies/mL was observed reaching a plateau at 32 hrs pi (Figure 3A). However, in PRRSV infected and App culture supernatant treated PAM cells, no increase in (+) gRNA copies/mL was detected. Moreover, in the presence of App culture supernatant, a significant decrease of (+) gRNA copies/mL was observed from 28 to 52 hrs pi compared to 4 hrs pi. Similarly, PRRSV sg mRNAs copies/mL began to rise between 8 and 16 hrs pi in PRRSV infected cells, reaching a plateau at 32 hrs pi while no increase of PRRSV sg mRNAs copies/mL was observed in the presence of App culture supernatant. A statistically significant decrease in sg mRNAs copies/mL was observed from 24 to 52 hrs pi, when compared with data at 4 hrs pi, in PRRSV infected and App culture supernatant treated cells (Figure 3B). Similarly to PAM cells, an increase of (+) gRNA and sg mRNAs copies/mL in PRRSV infected SJPL cells was observed between 8 and 16 hrs pi while no increase was observed in PRRSV infected cells treated with
App culture supernatant (Figures 3C and 3D). However, a significant increase of PRRSV (+)
gRNA and sg mRNAs copies/mL was observed in the presence of the bacterial supernatant in (+) gRNA and sg mRNA copies/mL from 38 to 52 hrs pi and from 24 to 52 hrs pi, respectively, when compared to the data at 4 hrs pi. Still, this increase was significantly lower compared to 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330
effect is less efficient in PRRSV infected SJPL cells compared to infected PAM cells (Figure 3). In MARC-145 cells, no significant difference was obtained between PRRSV infected cells treated or not with the App culture supernatant in both genome replication and transcription assays (Figures 3E and 3F). This latest result was not surprizing because it has been previously reported that App culture supernatant is not able to inhibit the replication of PRRSV IAF-Klop strain in infected MARC-145 cells [9, 24]. The early time pi results clearly indicate that App culture supernatant inhibits PRRSV infection before the first cycle of PRRSV genome replication/transcription in both PAM and SJPL cells. In addition, similar results were obtained using both infections protocols (data not shown).
Actin cytoskeleton modulation in App culture supernatant treated cells
App has been previously shown to degrade actin in vitro [32] and PRRSV needs an intact actin
cytoskeleton for cell infection and replication [5, 28]. To confirm the involvement of actin in the
App culture supernatant antiviral effect, the β-actin mRNA transcription level and protein
expression were determined in treated cells at 52 hrs pi. As shown in Figures 4A and 4C, the β-actin mRNA relative expression was lower in App culture supernatant treated PAM and MARC-145 cells compared to control untreated cells. In addition, the β-actin mRNA relative expression was lower in PRRSV infected and App culture supernatant treated PAM cells compared to control untreated cells (Figure 4A). Subsequently, the β-actin protein expression level was slightly decreased in App culture supernatant treated PAM cells compared to control untreated cells (Figure 4D). In SJPL cells, no modulation of β-actin mRNA and protein expression levels were observed (Figures 4B and 4E). Those results indicate that App culture supernatant modulation of the -actin expression seems to be cell type dependent. Then, the polymerization 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354
status of actin (i.e. filamentous actin: F-actin) was evaluated since its polymerization state is part of an intact cytoskeleton. A decrease in F-actin fluorescence intensity was observed in App culture supernatant treated PAM cells , but this decreasing was more pronounce in PRRSV infected cells treated with App culture supernatant (Figures 5A and 5B). In SJPL cells, immunofluorescence (IFA) revealed also a decrease in F-actin fluorescence intensity in App culture supernatant treated cells compared to mock untreated cells (Figure 5C). Interestingly, no marked difference was detected in regards to F-actin expression level between App culture supernatant treated and untreated MARC-145 cells.
IFA results showed a decrease in F-actin level in App culture supernatant treated cells, suggesting that actin is depolymerized. Moreover, our previous results obtained with a commercial antibodies microarray (Kinexus KAM-850) indicated that App culture supernatant modulates cofilin expression level in PAM treated cells (Table S1). Cofilin active form (i.e. dephosphorylated) is known to provoke F-actin depolymerization. Thus, it was important to determine the amount of total cofilin versus phosphorylated cofilin (P-cofilin) in App culture supernatant treated and PRRSV infected cell lines. Western blot analyses revealed that the total cofilin relative density in PRRSV infected PAM cells treated with App culture supernatant was higher compared to PRRSV infected cells alone, to App culture supernatant treated cells and to negative cells (Figure 6A). Interestingly, the relative density of P-cofilin was lower in infected PAM cells treated with the bacterial supernatant and also in PRRSV infected cells compared to the other experimental groups (Figure 6A). Since total cofilin was significantly increased and P-cofilin was significantly lower in infected PAM cells treated with the bacterial culture 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376
experimental group compared to other groups. In SJPL cells, there was a slight increase in total cofilin protein level in App culture supernatant treated cells compared to the other treatments (Figure 6B). No difference between control SJPL cells, PRRSV infected SJPL cells alone and
App culture supernatant treated SJPL infected cells were observed, which may explain why the App culture supernatant antiviral effect seems lower in SJPL cells compared to PAM cells
(Figure 6B). However, like in PAM cells, P-cofilin levels were lower in infected SJPL cells treated with the bacterial culture supernatant and also in PRRSV infected cells alone compared to the other experimental groups while higher levels were observed in SJPL cells treated with the bacterial culture supernatant compared to mock treated SJPL cells (Figure 6B). In MARC-145 cells, the total cofilin protein levels slightly increased only in PRRSV infected cells (Figure 6C). In a surprising way, it was observed that in infected MARC-145 cells treated with App culture supernatant there was more P-cofilin than in the other experimental groups, which may explain why App culture supernatant do not have an antiviral effect against PRRSV in infected MARC-145 cells.
Infectious viral particles production in PRRSV-infected cells treated with Cytochalasin D
The Cytochalasin D microfilament disrupting compound effect on PRRSV infection in PAM and MARC-145 cells is already known [5, 28]. However, it was important to test its efficiency in our experimental conditions and on cells that have not been previously tested, i.e. the SJPL cell line. For this purpose, all cell lines were infected and treated with 3 µM of Cytochalasin D using both infection protocols. The number of infectious virions in infected PAM cells in the presence of Cytochalasin D (102.2 TCID
50/mL) was significantly lower than in non-treated infected cells (105.5 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399
TCID50/mL) (P<0.05) (Figure 7A). Cytochalasin D completely blocked PRRSV infection in SJPL cells with at least a 500,000 times infectious titer reduction (P<0.05) (Figure 7B). Cytochalasin D was not able to inhibit PRRSV infection in MARC-145 cells (Figure 7C). Noteworthy, in PAM and SJPL cells, there is a complete inhibition of PRRSV infection in the presence of Cytochalasin D because the number of infectious virions obtained in infected cells treated with this compound was lower or equal to the infectious PRRSV particles measured at 4 hrs pi, which is considered to be the number of particles attached and/or entered into the cells (Figures 7A and 7B). Noteworthy, similar results were obtained with both infection protocols, i.e. with Cytochalasin D added 2 hrs prior or 4 hrs after PRRSV infection (data not shown). These results clearly demonstrate that Cytochalasin D, like App culture supernatant, inhibits PRRSV infection in PAM and SJPL cells, but not in MARC-145 cells.
App culture supernatant antiviral effect against different PRRSV strains
Results demonstrated that the number of infectious virions in LV PRRSV strain infected cells treated with App culture supernatant was significantly lower (107 to 1202 times lower) than in LV infected cells alone in all cell types (P<0.05) (Figure 8). Viral titers obtained with FMV13-LMIVV PRRSV strain demonstrated that the bacterial culture supernatant significantly reduces the number of infectious virions compared to FMV13-LMIVV infected cells alone (P<0.05) (Figure 8). Nonetheless, the App culture supernatant antiviral power against the FMV13-LMIVV strain in PAM cells was the smallest, the amount of PRRSV was 13.49 times lower when PRRSV infected cells were treated with App culture supernatant (Figure 8A). Viral titers obtained with FMV09-1155278 PRRSV strain demonstrated that App culture supernatant significantly reduces the number of infectious virions compared to infected cells alone (P<0.05) 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422
(Figure 8). Overall, the App culture supernatant antiviral efficacy varied between virus strains and between infected cells models. Noteworthy, the App culture supernatant antiviral effect tends to be lower in infected MARC-145 cells compared to the two other cell models (Figure 8D). 423
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DISCUSSION
The newly discovered antiviral activity of App culture supernatant against PRRSV was shown to be effective in the SJPL and PAM cells but not in MARC-145 cells [24]. It was also previously demonstrated that other viruses such as equine herpes virus type 1, swine influenza H1N1 and H3N2 infection could be inhibited by the App culture supernatant in the SJPL cells but to a significant much lower extent compared to PRRSV. Interestingly, the bovine adenovirus 3, bovine herpes virus type 1 and bovine viral diarrhea virus type 1 infections were not affected by the App culture supernatant, indicating that SJPL cells are metabolically active and able to support the growth of several other viruses. Since the impact of App culture supernatant on PAM cells viability and mortality was unknown, it was important to establish the PAM cells status following App culture supernatant treatment. Results indicate that the bacterial supernatant did not induce cell death since similar results were obtained between the control untreated and App culture supernatant treated cells (Figure 1). In addition, it was observed that in PRRSV infected cells treated with App culture supernatant antiviral, there was a significant increase in cell survival and a significant decrease in mortality rate compared to App culture supernatant non-treated PRRSV infected cells. This PAM cells survival increase can be the direct consequence of the lower PRRSV replication induced by the antiviral effect of App culture supernatant. Taken together, these results demonstrated that App culture supernatant treated PAM cells are viable, metabolically active and that the observed antiviral effect of the bacterial supernatant is not due to cell mortality.
In order to identify the App culture supernatant antiviral mechanism, the mRNA expression of type I and II IFN was determined because these cytokines are very important for the host 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448
strategies to evade their antiviral effects [35-38]. It was observed that App culture supernatant induces a basal or slight increased mRNA relative expression of IFN-γ and IFN- compared to untreated cells (Figure 2), indicating that App culture supernatant antiviral effect is not mediated via the induction of those cytokines. Lévesque and collaborators also observed that the bacterial supernatant did not induce type I IFN expressions in the SJPL cell line [24]. However, an induction of type II IFN expression was detected in SJPL cells, which suggest that App culture supernatant antiviral effect might be via the induction of IFN-γ but no modulation of IFN- mRNA expression was observed in App culture supernatant treated PAM cells. These results suggest that App culture supernatant antiviral effect may occur via other mechanisms that are cell type dependent. Moreover, it was demonstrated that mRNA relative expression of type I IFN was decreased in PRRSV infected PAM cells treated with the bacterial supernatant compared to PRRSV infected cells alone. Type I IFN mRNA decrease can be the direct effect of PRRSV replication inhibition by the bacterial antiviral effect or either by a component within the App culture supernatant. Noteworthy, RNA-Seq experiments will soon be carried out to better establish the impact of App culture supernatant over the modulation of cell mRNAs expression.
All stages of virus replication cycle are dependent on host cell machinery. For instance, 1) PRRSV entry occurs via receptor-mediated endocytosis and this process was demonstrated to be microfilament dependent [5, 28]; 2) PRRSV uncoating is known to be dependent on acidic pH of the early endosomes and also involved cellular proteases [5, 39, 40]; and 3) PRRSV genome replication/transcription is believe to be produced in autophagosome-like double-membrane vesicles [41, 42]. In order to identify at which PRRSV replication cycle step the bacterial antiviral effect occurs, the PRRSV genome replication and transcription were evaluated. Results clearly demonstrate that App culture supernatant antiviral effect against PRRSV takes place prior 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472
the first cycle of genome replication and transcription. The fact that during the first 4 to 8 hrs pi similar results were obtained using both PRRSV infection protocols (Figure 3), indicated that at least PRRSV attachment to cells is not inhibited by App culture supernatant treatment because it’s well known that this process in PAM reaches a maximum at one hour pi [5]. Overall, the results clearly indicate that App culture supernatant antiviral effect against PRRSV take place at least during the entry, uncoating or during the formation of the replication/transcription complex. At the moment, it is not possible to specify at which of these three virus replication steps the App culture supernatant antiviral effect occurs. Otherwise, the App culture supernatant antiviral effect seems more efficient in PAM cells compared to SJPL cells. The gRNA replication was entirely inhibited in PAM cells whereas, in SJPL cells, a small but statistically significant increase of (+) gRNA and sg mRNAs copies was observed over time (Figure 3) suggesting that in SJPL cells, few PRRSV particles can achieve a complete replication cycle. These findings can be explained because both cell types are phenotypically different. In fact, a recent study performed by Provost and colleagues has demonstrated that PRRSV receptors contain in both cell types are different [9]. Further studies should be conducted in order to identify PRRSV entry mediators and to investigate in detail PRRSV replication cycle in the new SJPL cells infection model. At the moment, there is no data to explain why MARC-145 cellular response in regards to App culture supernatant antiviral action is different. The major difference known between MARC-145 and PAM cells in regards to PRRSV replication cycle is the virus entry into the cell. PRRSV entry mediators in PAM and MARC-145 cells are different, confirming that virus entry differs between the two cell types. For instance, in MARC-145 cells the sialoadhesin is absent [6] and the sialic acids present in the virion are not essential for infectivity [43]. It was also reported that cholesterol is critical for PRRSV entry in MARC-145 cells and also suggest that PRRSV entry 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495
could be via a lipid-raft-dependent endocytosis [44, 45]. Therefore, the MARC-145 cells adapted IAF-Klop strain can use probably a completely different entry mechanism in both cell types, which makes PRRSV IAF-Klop strain resistant to App culture supernatant antiviral effect in MARC-145 cells. However, this observation might be strain specific since the results in Figure 8C show that the App culture supernatant possesses an antiviral activity in MARC-145 cells against three others genetically different PRRSV strains. More experiments are needed to elucidate this virus strain effect.
Garcia-Cuellar and colleagues have demonstrated that an App secreted 24kDa Zn-metalloprotease is able to degrade actin protein in vitro [32]. Moreover, several reports have revealed the important role of actin cytoskeleton on PRRSV infection [5, 6, 28, 46]. Therefore, based on these previous findings, it is more likely that App culture supernatant antiviral effect could be modulated through actin cytoskeleton. Interestingly, an antibodies microarray (Kinexus) revealed that cofilin 1 and LIMK1 (proteins involved in actin pathway) were modulated by PRRSV and App culture supernatant (Table S1). Consequently, the actin cytoskeleton modulation was investigated. It has been established that cofilin severing activity induces F-actin free ends accessible for actin polymerization and depolymerization [47, 48]. Cofilin has two statuses: unphosphorylated and phosphorylated. Its active form (i.e. unphosphorylated cofilin) is able to bind F-actin and promote its depolymerization [47]. LIMK is assumed to deactivate cofilin through its phosphorylation [48, 49]. Actin cytoskeleton was demonstrated to be involved in many RNA and DNA viruses’ replication cycle [50-53]. As an example, human immunodeficiency virus (HIV) has been shown to decrease P-cofilin [54]. It was demonstrated that cofilin activation and actin dynamics are very important for post-entry process of HIV of resting T cells. Moreover, Yoder et al. (2008) have reported that it was the attachment of HIV to 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518
the cell surface of the chemokine coreceptor CXCR4 that subsequently initiate the signaling toward cofilin activation and actin depolymerization. This process allows the resting T cells to be permissive to HIV infection. Therefore, a similar mechanism may also be involved in PRRSV post entry process since our data indicated that PRRSV IAF-Klop strain has induced a significant decrease of P-cofilin in all tested three cell types (Figure 6). In fact, during PRRSV infection, the microfilaments are a critical component necessary for PRRSV primary and secondary infection [5, 28]. Interestingly, there was more active cofilin in App culture supernatant treated infected PAM cells compared to other experimental groups (Figure 6A). This finding suggested a possible F-actin modulation, which was confirmed by IFA. The confocal microscopy revealed that F-actin fluorescence intensity was decreased in App culture supernatant treated cells, but its decrease was more pronounced in App culture supernatant treated infected PAM cells (Figures 5A and 5B). A previous report suggests that a negative correlation exists between the F-actin expression level and PRRSV infection [28], indicating that PRRSV decreases F-actin to favor its infection. Therefore, it is possible that PRRSV modulates the amount of F-actin that is needed for its infection but when certain low and high thresholds are exceeded, PRRSV infection is compromised. For that reason, it is suggesting that when PRRSV infected cells are treated with App culture supernatant, there is an increase of active cofilin that will subsequently induce F-actin depolymerization and thereafter PRRSV infection inhibition. Interestingly, a decrease of P-cofilin combined with a reduction of F-actin in App culture supernatant treated SJPL cells suggests that the antiviral mechanism in both PAM and SJPL cells might be similar at some points. In PRRSV infected MARC-145 cells treated with the bacterial antiviral, more P-cofilin (inactive form) was detected compared to other experimental groups (Figure 6). In addition, there was no F-actin depolymerization in the presence of App culture 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541
supernatant in MARC-145 cells (Figure 5C). Therefore, since App culture supernatant cellular response in MARC-145 cells differs from PAM and SJPL cells, these results highly suggest that
App culture supernatant cellular target is cofilin. Moreover, the App culture supernatant different
effects observed between treated cell lines could be explained by the cofilin/-actin ratio as previously reported by Bamburg and Bernstein (2010). The cofilin/-actin ratio plays a major role in the polymerization state of F-actin [55]. In fact, low cofilin/-actin ratios lead to F-actin polymerization and high cofilin/-actin ratios promote depolymerization of F-actin. Combination of Figure 4 and 6 western blot results show that in control PAM cells, the cofilin/-actin ratio was relatively low (0.53, data not shown), thus F-actin was polymerized. In App culture supernatant treated PAM cells, the cofilin/-actin ratio was slightly higher (0.94, data not shown) and therefore some F-actin was depolymerized compared to mock treated cells. In PRRSV infected PAM cells, the cofilin/-actin ratio was similar to App culture supernatant treated cells and F-actin was also partially depolymerized. In App culture supernatant and PRRSV infected PAM cells, the cofilin/-actin ratio was relatively higher (1.23, data not shown) compared to all other treatments, thus F-actin was found to be even more depolymerized as shown in Figures 5A and 5B. In SJPL cells, the ratio cofilin/-actin in App culture supernatant treated cells (1.21, data not shown) was higher compared to mock treated cells (1.03, data not shown) and this correlates with our observation of lower levels of F-actin polymerized (Figure 5C). In MARC-145 cells, cofilin/-actin ratios were similar between untreated and App culture supernatant treated cells, supporting also the observation that the same polymerization state of F-actin was observed between these two experimental groups as shown in (Figure 5C). Furthermore, those results suggest that the F-actin depolymerization phenomenon in PAM and SJPL cells was not related to the metalloprotease secreted by App that degrades β-actin in vitro [32], but rather to cofilin [48]. 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564
The different response that was observed between PAM and MARC-145 cells could be that inflammasome might be involve in the App culture supernatant effect. Inflammasomes are receptors and sensors proteins from the innate immune system that regulate the activation of caspase-1 and induce inflammation in response to infectious microbes, such as bacteria and viruses [56]. In macrophages, PRRSV infection has been demonstrated to induce capsase-1 and inflammasome activation [57, 58]. Moreover, it was shown that actin polymerization state also plays a major role in inflammasome activation [59]. It was also proposed that disruption of F-actin polymerization might activate the inflammasome [60]. Since no modulation in F-F-actin was observed in App culture supernatant treated MARC-145 cells (Figure 5C) and an important change in actin polymerization state was observed in App culture supernatant treated PAM and SJPL cells (Figure 5), then it could suggest the involvement of caspase-1 and inflammasome into the App culture supernatant antiviral effect. However, other experiments are needed to be conducted to confirm this hypothesis.
In order to confirm the involvement of actin cytoskeleton in App culture supernatant antiviral effect, the effect of Cytochalasin D (a drug that destabilizes actin filaments) on PRRSV replication was determined. The use of this drug has been a valuable tool for investigating the roles of actin filaments in cellular processes and in viral pathogenesis [5, 28, 53, 61-65]. Cytochalasin D was able to inhibit PRRSV replication in PAM and SJPL cells, but not in MARC-145 cells, such as App culture supernatant (Figure 7). In this study, Cytochalasin D was added few hours before or shortly after PRRSV infection and like App culture supernatant, it was able to inhibit PRRSV replication indicating that PRRSV was able at least to attach to the target cells (data not shown). Therefore, these results highly suggest that App culture supernatant probably inhibits PRRSV infection during its entry via clathrin-mediated endocytosis, since it is 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586
well known that this process is actin cytoskeleton dependent [5, 28, 66-68]. Conflicting data have been previously reported in regards to the antiviral effect of Cytochalasin D against PRRSV in infected MARC-145 cells. In fact, Cafruny and collaborators have demonstrated that Cytochalasin D at 1-2 µM concentration was able to inhibit PRRSV primary infection in MARC-145 cells [28]. However, in the present study, a higher dose of Cytochalasin D (3 µM) was used, and with this experimental condition, the PRRSV replication was not inhibited in infected MARC-145 cells. Noteworthy, this discrepancy can also be the consequence of having used different PRRSV strains in each study. Several studies have demonstrated that PRRSV isolates adaptation process in MARC-145 cells generate genetic changes, including deletions, insertions or substitutions and is characterized by higher titers, faster growth kinetics that make the newly adapted isolates less virulent than the wild type [69-73]. Then, it is possible that PRRSV IAF-Klop strain can use an entry mechanism that is actin cytoskeleton independent, in order to successfully replicate in the MARC-145 cells, which perfectly explains why the App culture supernatant antiviral effect is inefficient in MARC-145 cells (Figure 8C). Following this hypothesis, the App culture supernatant antiviral effect against other PRRSV strains was investigated. Interestingly, the replication of three PRRSV strains, which are classified within two different species [1], was significantly inhibited by App culture supernatant whereas no antiviral effect was detected against IAF-Klop strain in this cell model, indicating that the efficiency of the App culture supernatant antiviral effect is strain dependent in MARC-145 cells (Figure 8). This dependence can be due to differences in PRRSV strains entry mechanisms in MARC-145 cells as previously commented. Further studies need to be conducted in order to ascertain this hypothesis. Noteworthy, the App culture supernatant antiviral efficiency tends to be lower in infected MARC-145 cells compared to infected PAM and SJPL cells (Figure 8D). 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610
Perhaps because PRRSV strains used in this study are highly adapted to replicate in MARC-145 cells (i.e. this cell line is used for virus stock production and three out of the four PRRSV strains were isolated from clinical samples using this cell line).
In conclusion, this study clearly demonstrated that App culture supernatant inhibits PRRSV infection prior the first cycle of PRRSV genome replication/transcription in PAM and SJPL cells, probably via the activation of cofilin, which can provoke actin depolymerization and subsequently this phenomenon might affect PRRSV endocytosis. Further studies are in progress in order 1) to confirm that App culture supernatant affects PRRSV entry by endocytosis in PAM cells and 2) to find the active metabolite(s) present in App culture supernatant that is responsible for the antiviral effect.
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FUNDING INFORMATION
This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) discovery grants (to CAG and MJ) and Fonds de recherche du Québec – Nature et technologies (FRQNT) team research project program (to CAG and MJ). C. Provost was a recipient of a postdoctoral fellowship from the Canadian Swine Health Board. C.K. Traesel was a recipient of postdoctoral fellowships from Brazilian Conselho Nacional de Desenvolvimento
Científico e Tecnológico and FRQNT. Y. Hernandez Reyes was a recipient of a NSERC–
Alexander Graham Bell Canada Scholarship.
CONFLICTS OF INTEREST
All the authors have no conflict of interest related to the data of this manuscript.
ETHICAL STATEMENT
The animals (2 to 14 week old pigs) were humanely sacrificed following the ethic protocol 12-Rech-1640. This protocol was approved by Université de Montréal ethic committee, which is following the guidelines of the Canadian Council of Animal Care.
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